Method of treating crural or decubitus ulcer with zinc hyaluroric acid complex

ABSTRACT

Complexes of deprotonated hyaluronic acid with 3d metal ions of the 4th period of the Periodic Table and compositions containing these complexes as active ingredients or carriers. A process for the preparation of the complexes and compositions (pharmaceutical and cosmetic compositions) containing these complexes as active ingredients are disclosed in which zinc hyaluronate is preferably used as active ingredient.

This is a continuation of application Ser. No. 07/602,326, filed on 21Nov. 1990, now abandoned.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a National Phase application of PCT/HU90/00013 filed20 Feb. 1990 and based upon Hungarian national application Ser. No.891/89 of 24 Feb. 1989 under the International Convention.

FIELD OF THE INVENTION

The invention relates to novel associates (complexes) of deprotonatedhyaluronic acid with 3d metal ions of the 4th period of the PeriodicTable and compositions containing these associates (complexes) as activeingredients.

The invention further relates to a process for the preparation of thesenovel associates (complexes) and compositions containing theseassociates (complexes) as active ingredients.

According to a particularly preferred embodiment of the process of thepresent invention the aqueous solutions containing the novel associatesof deprotonated hyaluronic acid with 3d metal ions of the 4th period ofthe periodic table are directly prepared from an aqueous solution ofsodium hyaluronate.

The novel associates according to the present invention mainly involvezinc and cobalt hyaluronate. The compositions containing these latterassociates may be pharmaceutical (therapeutical) or cosmetic andoptionally other compositions. The compositions containing the novelassociates according to the invention are therapeutically effective fore.g.: the acceleration of epithelization of epithelium-deficient bodysurfaces; healing of crural ulcers, decubitus (bed-ulcers), primarilynot healing wounds, burns, radiation- or heat-induced wounds, vulgaracne and conglobated acnes, although they can be used in other areas,too.

BACKGROUND OF THE INVENTION

Hyaluronic acid is a macromolecule known for more than fifty years whichhas first been described by Meyer et al. [J. Biol. Chem. 107,629 (1954);J. Biol. Chem. 114, 689 (1936)]. The structure determination wasperformed by Weissman et al. [J. Am. Chem. Soc. 76, 1753 (1954)].Hyaluronic acid is a highly viscous native glucosaminoglycan containingalternating β₁₋₃ glucoronic acid and β₁₋₄ glucosamine moieties; itsmolecular weight is between 50000 and several (8 to 13) millions. Therecovery of hyaluronic acid is an task. The separation and use of anextrapure hyaluronic acid are described e.g. in the U.S. Pat. Nos.4,141,973 and 4,303,676 and in the European patent No. 0 144 019. Untilrecently hyaluronic acid has been employed as sodium salt e.g. intherapy, mainly in the ophithalmology surgery and cosmetics. The saltsof hyaluronic acid formed with alkaline, alkaline earth, magnesium,aluminum, ammonium or substituted ammonium ions may serve as carriersfor promoting the absorption of drugs (see the Belgian patentspecification No. 904,547). Heavy metal salts of hyaluronic acid(wherein "heavy metals" mean the elements of the 5th, 6th and 7thperiods of the Periodic Table as well as the lanthanides and actinides)and within these the silver salt are utilized as fungicidal agentswhereas the gold salt is employed for the treatment of arthritis (seethe patent specification WO 87/05517).

It has been proven by various structure-elucidating methods that thesecondary structure, i.e. the conformation of hyaluronic acid is changedby binding metal ions [W. T. Winter and A. Souther: J. Mol. Biol.517,761 (1977); J. K. Sheehan and E. D. T. Arkins: Int. J. Biol.Macromol. 5, 215 (1983); and N. Figueroa and B. Chakrabarti: Biopolymers17, 2415 (1978)]. Significantly varying effects on the molecularstructure can be exerted even by metal ions of similar character asshown by comparative X-ray study of potassium and sodium hyaluronate (A.K. Mitra et al.: J. Macromol., Sci. Phys. 824, 1 and 21 (1985)7. This isall the more valid for compounds of hyaluronic acid formed with metalions of various sorts bearing various charges.

No reference relating to associates (complexes) of hyaluronic acidformed with 3d metal ions of the 4th period of the Periodic Table can befound in the literature; actually, according to gel filtrationchromatography examinations, hyaluronic acid, in contrast with toheparin, is unable to bind zinc ions (R. F. Parish and W. R. Fair:Biochem J. 193,407-410 (1981)7.

In spite of the fact that, according to the literature, hyaluronic acid(or its sodium salt) is unable to bind zinc ions, we undertook toinvestigate the coordination chemistry of the interaction betweenhyaluronic acid and 3d metal ions of the 4th period of the PeriodicTable and among these, chiefly, zinc and cobalt ions. Since hyaluronicacid is nearly exclusively commercialized as its sodium salt thus beingthe basic substance of all systems containing hyaluronate, ourinvestigations were begun on the interaction of sodium ions andhyaluronate. For this purpose the free sodium ion activity of aqueoussodium hyaluronate solutions was measured by using a sodium selectiveglass electrode. It was unambiguously found from these measurements thatnot more than 60% of sodium ions introduced as equivalent together withthe carboxylate groups of hyaluronate are present as free ions in theaqueous solutions whereas the remainder of 40% is in a form bound to thehyaluronate.

According to our measurements, by increasing the sodium ionconcentration the amount of the sodium ions bound can be raised to 50-55% calculated for all available carboxylate groups. Thus, it has beenverified that, as contrasted with the common properties of salts, sodiumhyaluronate is not completely dissociated in aqueous solution.

DESCRIPTION OF THE INVENTION

In the next step of our investigations an aqueous solution of sodiumhyaluronate was titrated with zinc chloride solution by using a sodiumion-selective electrode as mentioned above for following the change inthe activity of free sodium ions in the system. A characteristic curvereflecting the process is shown in FIG. 1. It is perceivable that sodiumions originally bound to hyaluronate are liberated on the effect of zincions. Based on the results of these measurements the total sodium ionconcentration is liberated by an equivalent amount of zinc, a factunequivocally proving that zinc ions are more strongly bound tohyaluronate than are sodium ions. Thus, the earlier statement thathyaluronic acid would be unable to bind zinc ions [R. F. Parrish and W.R. Fair: Biochem. J. 193, 407 (1981)] has experimentally been refuted.

Thereby, previously held by workers skilled in the art was disproved.

From our investigations discussed above it became clear that, throughthe interaction of equivalent amounts of sodium hyaluronate and zincions (zinc chloride) in aqueous solution a zinc hyaluronate associatewith a stoichiometric composition is formed. After an appropriateisotonization the solution obtained can directly be used fortherapeutical purposes and the zinc compound need not to be prepared insolid state in a separate process. Preliminary examinations carried outby using cobalt ion and other 3d metal ions led to similar results.

Nevertheless, the complex was prepared in solid state forcharacterization and the direct environment of the zinc ion wasdetermined by using the `Extended X-ray Absorption Fine Structure"(EXAFS) method. It has been found that zinc is surrounded by four oxygenatoms in the first coordination sphere. The length of the Zn-O bonddistances is 199 pm whereas two carbon atoms are present in a longerdistance of 241 pm from the zinc atom.

According to our examinations zinc hyaluronate significently differsfrom the analogous copper complex which latter contains four equatorialand two axial Cu-O bonds with the values of 194 and 234 pm,respectively. The distance between the copper atom and the next twocarbon atoms is 258 pm. The structure of the cobalt complex is similarto the zinc complex but not to the copper complex.

Thus, the present invention relates to associates (complexes) ofdeprotonated hyaluronic acid with 3d metal ions of the 4th period of thePeriodic Table.

The invention further relates to a composition containing as activeingredient or carrier an associate (complex) of hyaluronic acid with 3dmetal ions of the 4th period of the Periodic Table, optionally inadmixture with other active ingredients and/or additives.

According to another aspect of the invention there is provided a processfor the preparation of the novel associates (complexes) of theinvention, which comprises

a) adding an aqueous solution containing the equivalent amount of asalt, preferably the chloride of one of 3d metal ions of the 4th periodof the periodic table to an aqueous solution of sodium hyaluronate or toan other salt (alkaline or alkaline earth metal salt, optionally silversalt) of hyaluronate; or

b) dissolving an associate formed from hyaluronic acid with a quaternaryammonium salt in an aqueous suspension in a solvent couple containingthe aqueous solution of a 3d metal ion of the 4th period of the PeriodicTable and a solvent which is partially miscible with water, preferablyn-butanol; then

precipitating the associate (complex) obtained of hyaluronic acid withthe 3d metal ion of the 4th period of the Periodic Table by an alkanolor alkanone in a known manner, or

separating the precipitate from the solution and then, if desired

drying it under mild conditions.

This process serves for the preparation of aqueous solutions containingas active ingredient a zinc hyaluronate associate (complex) or a similarassociate of a 3d metal ion of the 4th period of the Periodic Table,respectively. These solutions were in each case prepared by the directreaction of the metal ion with the hyaluronate component. This method ofpreparation made unnecessary to previously separate the activeingredients mentioned above in a solid state. In the solution preparedby using the process of the invention the amount of free (metal-unbound)hyaluronate is negligible even in the presence of an equivalent amountof zinc. In the presence of an excess of zinc ions the formation of thezinc hyaluronate associate (complex) becomes quantitative.

In the course of preparation of the metal associates as discussed abovethe pH remains at a value of about 5. In the case of a 0.2% byweight/volume (wt./vol.) hyaluronate solution the pH reaches a value of5.4 whereas in the case of 0.5 % by wt./vol. the pH value is 5. Whennecessary, the pH of the latter system can be adjusted to a value of 5.5to 5.6 by adding a few drops of an isotonic sodium acetate solution.

Solutions of two sorts containing zinc hyaluronate as active ingredienthave been prepared by using the process discussed above.

1. Zinc hyaluronate solution made isotonic by an excess of zincchloride:

Taking into consideration that free zinc chloride alone may alsopreferably be used in the dermatology, the osmotic pressure of the zinchyaluronate solution was adjusted to the isotonic value by using anexcess of zinc chloride. The solution thus obtained did not contain anyfree (zinc-unbound) hyaluronate at all but an excess of zinc chloridewas present in the system together with zinc hyaluronate.

2. Zinc hyaluronate solution made isotonic by a monosaccharide or asugar alcohol:

For a therapeutic use wherein the presence of hyaluronate-unbound zincions is not indicated, the solution containing zinc ions in an amountequivalent to the hyaluronate was made isotonic by using a polyalcohol(sugar alcohol, preferably sorbitol) or a mono- or disaccharide(preferably glucose). The free zinc ion and free hyaluronate content ofthese latter systems did not reach 5% of the total zinc or totalhyaluronate content, respectively.

In the course of utilizing the associates according to the inventionion-free compositions may eventually be required. Namely, the associatesprepared according to the above process of the invention usually containsodium chloride of an other salt formed from the starting hyaluronatecation and the anion of the 3d metal salt.

Two different process variants can be used for the preparation of asalt-free hyaluronic acid associate formed with a 3d metal ion. Theseare as follows.

a) A solution of a quaternary ammonium salt is portionwise added to thesolution of a known hyaluronate, preferably sodium hyaluronate. After asatisfying purification, the novel quaternary ammonium hyaluronateassociate precipitated is dissolved under vigorous stirring in a solventcouple consisting of an aqueous solution of a 3d metal ion of the 4thperiod of the Periodic Table and a solvent which is partially misciblewith water, preferably n-butanol. The two phases are allowed toseparate, then the hyaluronate associate is precipitated by adding analkanol or alkanone to the aqueous phase, the precipitate is separatedand washed; or

b) after adding 2.0 to 3 volumes of a C₁₋₃ alkanol or C₃₋₄ alkanoneunder stirring to a zinc hyaluronate solution, suitably to a notisotonized solution containing zinc chloride in an amount equivalent tothe hyaluronate, the zinc hyaluronate precipitated is filtered andwashed with the alkanol or alkanone, respectively used for theprecipitation. When necessary, the zinc hyaluronate is dissolved inion-free water and the precipitation is repeated.

When a solid ion-free zinc hyaluronate is needed, the precipitate isdried under reduced pressure under mild conditions. In the case of ademand on an ion-free zinc hyaluronate solution it is preferable todissolve the zinc hyaluronate made free from the solvent. According toany of both process variants an ion-free solid or dissolved product isobtained with an optional purity depending on the quality of thestarting zinc hyaluronate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a titration curve showing the titration of an aqueous sodiumhyaluronate solution with zinc chloride to form zinc hyaluronate andliberate sodium ion.

FIG. 2 is a series of two graphs showing the results of the clinicaltreatment of patients suffering from crural ulcers using zinchyaluronate according to the curve marked with a cross and using sodiumhyaluronate using the curve marked with a square. The x-axis shows theduration of treatment and the y-axis shows the severity of the disease.

FIG. 3 is a series of bar graphs showing the severity of crural ulcerstreated with sodium hyaluronate and zinc hyaluronate, respectively, overperiods of 1, 2, 3 and 4 weeks.

The results of the clinical-pharmacological investigation on acomposition (Example 13) containing as active ingredient the zinchyaluronate according to our invention are shown on a crural ulcertreatment used for the acceleration of epithelization ofepithelium-deficient surfaces. A composition containing sodiumhyaluronate was used as control.

This examination was carried out on 12 or 14 ulcers, respectively of 8or 12 patients suffering from crural ulcer. The distribution of thepatients of both group accoding to sex and age as well as to the natureof the disease was as follows.

    __________________________________________________________________________                                         Character of                             Active    No. of              No. of ulcers                                                                        the ulcer*                               ingredient                                                                              patients                                                                           Woman                                                                              Man                                                                              Average age                                                                          treated                                                                              A V  M                                   __________________________________________________________________________    Zinc hyaluronate                                                                        12   10   2  63.9   14     2 9  1                                   Sodium hyaluronate                                                                      8    6    2  65.7   12     --                                                                              7  1                                   __________________________________________________________________________     *A = arterial; V = venous; M = mixed                                     

The treatments were performed in such a way that before beginning thetreatment a purifying therapy was carried out according to the actualclinical state of the ulcer. The treatment with zinc or sodiumhyaluronate, respectively, was commenced on ulcers nearly purified or incases where a significant diminution of the sorbes was observed. Thetreatment was carried out daily once in such a way that the compositionwas dropped onto the surface of the purified ulcer in an amount wettingthe surface of the wound with a thin layer.

The composition was used for 4 weeks. At the beginning of the treatmentand then once in a week the data sheet was filled out and the ulcers ofthe patients were documented by photographs. A discharge sample wastaken for bacteriological examination.

The characteristics as well as the severity of the epithelial lesionswere marked with the following symbols and scores.

    ______________________________________                                        Characteristics        Severity                                               ______________________________________                                        Area (a)                                                                      0                      0                                                      Below 10 cm.sup.2      1                                                      Between 10 cm.sup.2 and 25 cm.sup.2                                                                  2                                                      Above 25 cm.sup.2      3                                                      Infectedness (b)                                                              Clinically pure        0                                                      Coated in 50%          1                                                      Coated in 100%         2                                                      Necrosis (c)                                                                  (only in the case of an arterial ulcer)                                       negative               0                                                      Below 10%              1                                                      Between 10% and 15%    2                                                      100%                   3                                                      No necrosis            4                                                      ______________________________________                                    

Evaluation

For evaluation the values of the separate characteristics weredetermined and the general severity score was calculated by using thefollowing formula: ##EQU1##

The results of the clinical pharmacological investigations areillustrated in FIG. 2. The results of the treatment with zinchyaluronate is shown on the curve marked with a cross whereas that ofthe treatment with sodium hyaluronate is illustrated on the curvedenoted with a square as a function of the number of weeks involving thetreatment. The score value plotted on the ordinate represents thegeneral severity index calculated by using the above formula.

For a more correct comparison of zinc hyaluronate to sodium hyaluronateused as control the relative correct values related to the startingscore values as 100% are illustrated in FIG. 3.

The change in the relative correct values was statistically evaluated asa function of number (1 to 4) of weeks. On the zinc and sodiumhyaluronate treatment, the number of ulcers decreased below a relativescore value of 90%, 80%, 70% and 60%, respectively after 1, 2, 3 and 4weeks was investigated. The results are summarized in Table 1.

                                      TABLE 1                                     __________________________________________________________________________              1st week                                                                              2nd week                                                                              3rd week                                                                              4th week                                    __________________________________________________________________________    Active ingredient                                                                       Distribution of the relative score value                            of the composition                                                                      90%     80%     70%     60%                                                   below                                                                             above                                                                             below                                                                             above                                                                             below                                                                             above                                                                             below                                                                             above                                   Zinc hyaluronate                                                                        12  2   11  3   11  3   11  3                                       Sodium hyaluronate                                                                      4   8   7   5   6   6   3   9                                       __________________________________________________________________________

It can be stated from Table 1 that the treatment with zinc hyaluronatewas in every week advantageous in comparison to the results obtainedwith sodium hyaluronate used as control.

The statistical analysis of the response obtained for the hypothesis inquestion proved that the advantage of the zinc hyaluronate compositionwas highly significant (p 99%) in comparison to sodium hyaluronate.

In a further statistical working-up, a more detailed destribution of therelative score values was investigated as a function of the time oftreatment. The results obtained are summarized in Table 2.

                  TABLE 2                                                         ______________________________________                                                  Number and score value of the ulcers                                Active ingredient                                                                         above    between   between below                                  of the composition                                                                        90%      90 and 70%                                                                              70 and 50%                                                                            50%                                    ______________________________________                                        1st week                                                                      Zinc hyaloronate                                                                          2        7         5       0                                      Sodium hyaluronate                                                                        8        3         0       1                                      2nd week                                                                      Zinc hyaluronate                                                                          0        6         7       1                                      Sodium hyaluronate                                                                        4        3         5       0                                      3rd week                                                                      Zinc hyaluronate                                                                          0        1         8       2                                      Sodium hyaluronate                                                                        2        5         5       1                                      4th week                                                                      Zinc hyaluronate                                                                          2        1         7       3                                      Sodium hyaluronate                                                                        1        5         3       3                                      ______________________________________                                    

The data of Table 2 similarly support the advantage of zinc hyaluronate.The more detailed statistical examinations show the significance todecrease depending on the time of treatment.

Summing up: on an evaluation of the clinical-pharmacologicalinvestigations the higher efficiency of zinc hyaluronate could be proveneven at a low number of ulcers; this advantage could particularly besupported in the starting period of the treatment.

The invention is illustrated in more detail by the following nonlimiting Examples.

SPECIFIC EXAMPLES

The protein content of hyaluronate (HA) was determined by using themethod of O. H. Lowry [J. Biol. Chem. 193 (1951)]; the viscosity ofhyaluronate was measured in an Oswald's viscometer in a physiologicalsaline solution at 25° C. The value of the intrinsic viscosityextrapolated to "0" concentration, i.e. ##STR1## is given below. The HAcontent was determined by using Bitter's method [Anal. Biochem 4, 330(1962)].

EXAMPLE 1

Preparation of a zinc hyaluronate solution

40.18 mg of sodium hyaluronate are dissolved in 20.0 ml of twicedistilled water. Thus, the starting concentration of hyaluronic acid is2.009 mg/ml, the equivalent concentration of the solution is 4.241×10 ⁻³mol/liter (Na⁺ or hyaluronic acid dimer unit). In the course of themeasurement, a zinc chloride solution of 0.05154 mol/liter concentrationis added to the reaction mixture through a microburet. The solution isfirst added in little portions (0.05 ml) and then in larger portions(0.1to 0.2 ml). The potential change in the solution is measured by using aprecision potentiometer with digital display and sodium ion-selectiveglass and silver/silver chloride electrodes. The titration is continueduntil the potential measured is not further changed by adding anadditional portion of the titrating solution. (The measuring system wascalibrated under conditions analogous to the practical measurement.)

The selectivity of the sodium ion-selective electrode was observed alsoin the presence of Zn²⁺ ions in order to control that the potentialchange in the practical measurement was caused by the liberated Na⁺ ionsand not the Zn²⁺ ions introduced to the solution. A 2.00×10⁻³ M sodiumchloride solution was titrated by using the zinc chloride titratingsolution under conditions similar to the above conditions. On increasingthe concentration of Zn²⁺ from 0 up to 4×10⁻³ mol/liter a potentialincrease of about 2 mV was observed whereas the practical measurementshowed a change of about 20 mV under similar conditions. Thus, theevaluation had no obstacle. In the course of measurement the increase inthe sodium ion activity calculated from the measurement data verifiedthe quantitative formation of the zinc associate.

Preparation of a zinc chloride solution

Since a solution containing zinc chloride in an accurate concentrationcannot be prepared by direct weighing-in, first a solution with thenearly desired concentration is prepared. On preparing this solution noacid should be used thus it may occur that the zinc chloride weighed inwill not completely be dissolved. After sedimentation of the insolubleresidue (about 30 minutes) the volumetric flask is filled up to the markand the solution is filtered through a filter paper.

The accurate concentration of the filtrate is determined bycomplexometric titration by using buffer 10 and eryochrom black-Tindicator. The zinc chloride solution with an accurate concentration of0.100 mol/liter is prepared by the precise dilution of this solution.

The characteristics of sodium hyaluronate used for the preparation ofsolution are as follows:

    ______________________________________                                        Molecular weight     1850000 daltons                                          Protein content      0.07% by wt.                                             UV absorption                                                                  ##STR2##            0.133                                                     ##STR3##            0.075                                                     ##STR4##            13.7 dl/g                                                HA.sup.x content     98.12% by wt.                                            ______________________________________                                         .sup.x HA = hyaluronic acid (as abbreviated herein)                      

EXAMPLE 2

Preparation of a solution for dermatologic and cosmetic use

12.5 ml of a zinc chloride solution of 0.100 mol/liter concentrationprepared with ion-free water are added to 0.50 g of sodium hyaluronateweighed in a 100 ml volumetric flask. (An other concentration of zincchloride may also be used but the amount of zinc chloride should be thesame.) Sodium hyaluronate is allowed to swell (for 12 hours) inthesolution filled up to the mark with ion-free water to obtain a zinchyaluronate solution of 0.5% by wt./vol.

The characteristics of sodium hyaluronate used for preparing the abovesolution are as follows:

    ______________________________________                                         ##STR5##            16,5 dl/g                                                Protein content      0.8% by wt.                                              ______________________________________                                    

EXAMPLE 3

Preparation of a zinc hyaluronate solution for use in injectablesolutions

The operations described in this Example are carried out under sterileconditions.

5.0 ml of a zinc chloride solution of 0.100 mol/liter concentrationprepared with twice distilled water (water for injection use,pyrogen-free, sterile) are added to 0.20 g of sodium hyaluronate (ofpure powder quality) weighed in a 100 ml volumetric flask, then thevolume is filled up to 50 ml with twice distilled water. Sodiumhyaluronate is allowed to swell overnight, then dissolved by shaking andthe solution filled up to the mark with twice distilled water. Thesolution obtained is filtered through a membrane filter (0.45μ poresize) to give a zinc hyaluronate solution of 0.24 by wt./vol.

The characteristics of the sodium hyaluronate used for preparing theabove solution are as follows:

    ______________________________________                                        Quality        pure, pyrogen-free sterile powder                              Molecular weight                                                                             1850000                                                        Protein content                                                                              0.07% by wt.                                                   UV absorption                                                                  ##STR6##      0.133                                                           ##STR7##      0.075                                                          HA content     98.12% by wt.                                                   ##STR8##      13.7 dl/g                                                      ______________________________________                                    

EXAMPLE 4

Preparation of an ion-free zinc hyaluronate solution

600 ml of ethanol of analytical grade are added under stirring to 200 mlof 0.50 % by wt./vol. zinc hyaluronate solution obtained according toExample 2, the precipitated zinc hyaluronate is filtered on a glassfilter, washed twice with 50 ml of ethanol each of the same quality andthen dried under reduced pressure. Thus, 0.88 g of zinc hyaluronate isobtained which is used for preparing a 0.50 % by wt./vol. zinchyaluronate solution in the way described in Example 2. The zinchyaluronate solution obtained does not contain any sodium chloridearising from the reaction between sodium hyaluronate and zinc chloride;thus, it is practically ion-free.

EXAMPLE 5

Preparation of ion-free zinc hyaluronate or its solution fortherapeutical use

The operations described in this Example are carried out under sterileconditions.

1500 ml of ethanol (purest quality) are portionwise added to 500 ml ofzinc hyaluronate solution prepared according to Example 3 understirring. After the addition the system is stirred for 30 minutes, thezinc hyaluronate precipitate is filtered on a glass filter, washed 3times with 100 ml of ethanol (purest quality) each and dried underreduced pressure under mild and sterile conditions.

EXAMPLE 6

Preparation of ion-free zinc hyaluronate

200 ml of 10% by wt. solution of Hyamine® 1622 (puriss)(benzyldimethyl}-2-[2-p-(1,1,3,3-tetramethylbutyl)penoxy]ethoxy}ethylammoniumchloride) are added under stirring to the solution containing 1 g ofsodium hyaluronate in 400 ml of twice distilled water. The precipitatei.e. the hyaluronic acid quaternary ammonium associate formed isseparated by centrifuging, washed twice with 100 ml of twice distilledwater each and again centrifuged. The washed precipitate is dissolved ina solvent couple consisting of 400 ml of 2% by wt./vol. zinc chloride inaqueous solution (pH 5.0 to 5.4) and 400 ml of n-butanol. After allowingto separate the two phases, the aqueous layer containing the dissolvedzinc hyaluronate is filtered through a membrane filter (0.45μ poresize), then zinc hyaluronate is precipitated by adding 3 volumes ofethanol, filtered on a glass filter, washed with ethanol and dried in anitrogen atmosphere under mild conditions to obtain 0.82 g of zinchyaluronate.

When necessary, a 0.50 % by wt./vol. solution is prepared from the zinchyaluronate obtained which is then further purified as described inExample 4. The characteristics of sodium hyaluronate used as startingmaterial are as follows:

    ______________________________________                                         ##STR9##           16.5 dl/g                                                 Protein content     018% by wt./vol.                                          ______________________________________                                    

Zinc hyaluronate can be prepared as described above also from associatesformed from other quaternary ammonium salts. Quaternary salts useful forthis purpose are e.g.:

a) carbotetradecyloxymethyl-trimethylammonium chloride (see theHungarian patent specification No. 188,537),

b) hexadecylpyridinium chloride,

c) cetylpyridinium chloride,

d) trimethylammonium chloride and the like.

EXAMPLE 7

Preparation of cobalt hyaluronate

The process described in Example 6 is followed, except that thehyaluronic acid quaternary ammonium associate is dissolved in a solventcouple consisting of a 2% by wt./vol. cobalt(II) chloride.6H₂ O aqueoussolution and n-butanol.

EXAMPLE 8

Preparation of an aqueous solution containing 0.50% by wt./vol. of zinchyaluronate made isotonic by zinc chloride

About 50 ml of a zinc chloride solution of 0.110 mol/liter concentrationare added to 0.50 g of sodium hyaluronate in a 100 ml volumetric flaskand then allowed to swell overnight. Then, the sodium hyaluronate isdissolved by shaking and the flask is filled up to the mark with a zincchloride solution of 0.110 mol/liter concentration.

The osmotic pressure of the solution obtained is 0.1491 mol/liter asexpressed in equivalent sodium chloride concentration, the value of pHis 5.0. When necessary, the pH value is adjusted to 5.5 to 5.6 by adding2.00 ml of a sodium acetate solution of 0.150 mol/liter concentration.After adjusting the pH value, the osmotic pressure of the solution is0.1489 as expressed in equivalent sodium chloride concentration.

The zinc hyaluronate solution is prepared from the particularly puresodium hyaluronate described in Example 3 with twice distilled waterunder aseptic conditions, then the solution is filtered through amembrane filter (0.45μ pore size).

The solution obtained can be used in injectable compositions, too.

EXAMPLE 9

Preparation of an aqueous solution containing 0.2% by wt./vol. of zinchyaloronate made isotonic by zinc chloride

For a final volume of 100 ml, 0.20 g of sodium hyaluronate is weighed inand dissolved in a zinc chloride solution of 0.120 mol/literconcentration.

The dissolution and preparation of the zinc chloride solution ofprecisely 0.120 mol/liter concentration are carried out according toExample 1 (according to the sense by changing the amount of zincchloride).

The osmotic pressure of the solution is 0.154 mol/liter as expressed inequivalent sodium chloride concentration; the pH shows a value of 5.3 to5.4.

    ______________________________________                                        HA content:         1.96 mg/ml                                                Viscosity:          15.9 dl/g                                                 Protein concent:    0.015 mg/ml                                               Purity of the solution.sup.x :                                                                    A.sub.660.sup.1 cm = 0.015                                ______________________________________                                         .sup.x Based on the absorbance measured at 660 nm in an 1 cm cuvet       

The solution is prepared by using the sodium hyaluronate of the qualitycharacterized in Example 2 and used first of all for the preparation ofdermatologic and cosmetic compositions.

EXAMPLE 10

Preparation of an aqueous solution containing 0.50 % by wt./vol. of zinchyaluronate made isotonic by glucose

The solution of this Example contains sodium hyaluronate and thecalculated equivalent amount of zinc chloride.

12.50 ml of a zinc chloride solution of 0.100 mol/liter concentrationare added to 0.50 g of sodium hyaluronate weighed in a 100 ml volumetriclask. (Another concentration of zinc chloride may also be used but theamount of zinc chloride should be the same.) Sodium hyaluronate isallowed to swell for 12 hours in the solution of zinc chloride filled upto 50 ml with ion-free water, then dissolved by shaking. Thereafter,24.50 ml of a glucose solution of 1.00 mol/liter concentration are addedand filled up to the mark with ion-free water.

The osmotic pressure of the solution is 0.1495 mol/liter as expressed inequivalent sodium chloride concentration; the pH shows a value of 5.4.Total zinc concentration=1.25×10⁻² mol/liter.

The solution is prepared by using the sodium hyaluronate of the qualitycharacterized in Example 2 and used first of all for the preparation ofdermatological and cosmetic compositions.

EXAMPLE 11

Preparation of an aqueous solution containing 0.2% by wt./vol. of zinchyaluronate made isotonic by glucose

The solution of this example contains sodium hyaluronate and thecalculated equivalent amount zinc chloride.

5.0 ml of a zinc chloride solution of 0.100 mol/liter concentration areadded to 0.20 g of sodium hyaluronate weighed in a 100 ml volumetricfalsk, then the volume is completed to 50 ml with deionized water. Afterallowing to swell overnight, sodium hyaluronate is dissolved by shaking,27.0 ml of a glucose solution of 1.00 mol/liter concentration are addedand the flask filled up to the mark with ion-free water.

The osmotic pressure of the solution is 0.151 mol/liter as expressed inequivalent sodium chloride concentration; the pH shows a value of 5.6 to5.7; Total zinc concentration=5×10⁻³ mol/liter.

EXAMPLE 12

Preparation of an aqueous solution containing 0.5% by wt./vol. of zinchyaluronate made isotonic by sorbitol

The zinc hyaluronate solution described hereinafter is prepared underaseptic conditions from sodium hyaluronate of particularly high puritydescribed in Example 3 and distilled water. The solution contains zincchloride in an equivalent amount calculated for sodium hyaluronate.

The process described in Example 10 is followed, except that, instead ofthe glucose solution, 23.50 ml of a sorbitol solution of 1.00 mol/literconcentration (182.19 g of D-sorbitol in 1 liter) are added to the zinchyaluronate solution.

The solution thus prepared is filtered through a membrane filter (0.45μpore size). This solution can be used for any purpose includinginjectable compositions.

The osmotic pressure of the solution is 0.1520 mol/liter as expressed inequivalent sodium chloride concentration; the pH shows a value of 5.5;Total zinc concentration=1.25×10⁻² mol/liter.

EXAMPLE 13

Preparation of an aqueous solution containing 0.2% by wt./vol. of zinchyaluronate made isotonic by sorbitol

The solution described in this Example contains zinc chloride in anequivalent amount calculated for sodium hyaluronate.

The zinc hyaluronate solution described hereinafter is prepared underaseptic conditions from sodium hyaluronate of particularly high puritydescribed in Example 3 with twice distilled water.

The process of Example 12 is followed, except that 0.2 g of sodiumhyaluronate is dissolved, 5 ml of zinc chloride solution of 0.100mol/liter concentration, then 26.50 ml of a sorbitol solution of 1mol/liter concentration are added and finally, the solution is filled upto 100 ml. The solution thus prepared is filtered through a membranefilter (0.45μ pore size). This solution can be used for any purposeincluding injectable compositions.

The osmotic pressure of the solution is 0.1501 mol/liter as expressed inequivalent sodium chloride concentration; the pH shows a value of 5.6;Total zinc concentration=5×10⁻³ mol/liter.

    ______________________________________                                        Hyaluronate content:                                                                              2.03 mg/ml                                                Viscosity:          16.1 dl/g                                                 Protein content:    0.016 mg/ml                                               Purity of the solution.sup.x :                                                                    A.sub.660.sup.1 cm = 0.010                                ______________________________________                                         .sup.x Based on the absorbance measured at 660 nm in an 1 cm cuvet       

EXAMPLES 14 TO 26

In the following Examples the components of various compositions(pharmaceutical and cosmetic compositions) are given in relation toformulation types selected by us. The preparation of zinc hyaluronatesolutions made isotonic are described in the preceding Examples Here,"distilled water for injection purpose" means twice distilled waterprepared under aseptic conditions.

I. Injectable solutions

Compositions of Examples 14 to 17 are used for intracutaneousadministration whereas that of Example 18 serves for intraocular use.The active ingredient of the quality described in Example 3 is employedin these Examples.

EXAMPLE 14

    ______________________________________                                        Zinc hyaluronate active ingredient                                                                       2.0    mg                                          Sorbitol                   48.3   mg                                          Final volume of the aqueous solution prepared                                                            1.0    ml                                          with distilled water for injection purpose                                    ______________________________________                                    

EXAMPLE 15

    ______________________________________                                        Zinc hyaluronate active ingredient                                                                       5.0    ml                                          Sorbitol                   42.8   mg                                          Final volume of the aqueous solution prepared                                                            1.0    ml                                          with distilled water for injection purpose                                    ______________________________________                                    

EXAMPLE 16

    ______________________________________                                        Zinc hyaluronate active ingredient                                                                       2.0    mg                                          Propyl p-hydroxybenzoate   0.05   mg                                          Methyl p-hydroxybenzoate   0.5    mg                                          Glucose                    48.6   mg                                          Final volume of the aqueous solution prepared                                                            1.0    ml                                          with distilled water for injection purpose                                    ______________________________________                                    

EXAMPLE 17

    ______________________________________                                        Zinc hyaluronate active ingredient                                                                       5.0    mg                                          Propyl p-hydroxybenzoate   0.05   mg                                          Methyl p-hydroxybenzoate   0.5    mg                                          Glucose                    44.1   mg                                          Final volume of the aqueous solution prepared                                                            1.8    ml                                          with distilled water for injection purpose                                    ______________________________________                                    

EXAMPLE 18

    ______________________________________                                        Zinc hyaluronate active ingredient                                                                       10.0   mg                                          Potassium sorbate          1.0    mg                                          Sorbitol                   41.0   mg                                          Final volume of the aqueous solution prepared                                                            1.0    ml                                          with distilled water for injection purpose                                    ______________________________________                                    

Compositions described in Examples 20 to 28 are mainly used fordermatologic and cosmetic purposes. The active ingredient of the qualitydescribed in Example 2 is employed in these Examples.

II. Solutions for topical use

EXAMPLE 19

    ______________________________________                                        Zinc hyaluronate active ingredient                                                                       5.0    mg                                          Potassium sorbate          1.0    mg                                          Sodium acetate             24.6   mg                                          Final volume of the aqueous solution prepared                                                            1.0    ml                                          with distilled water                                                          ______________________________________                                    

EXAMPLE 20

    ______________________________________                                        Zinc hyaluronate active ingredient                                                                       2.0    mg                                          Potassium sorbate          1.0    mg                                          Sorbitol                   48.3   mg                                          Final volume of the aqueous solution prepared                                                            1.0    ml                                          with distilled water                                                          ______________________________________                                    

III. Gels for topical use

EXAMPLE 21

    ______________________________________                                        Zinc hyaluronate active ingredient                                                                           20.0   mg                                      Acrylic acid polymerisate      200    mg                                      Sodium hydroxide of 30% concentration                                                                        50     mg                                      Potassium sorbate              10     mg                                      Distilled water         up to  10.0   mg                                      ______________________________________                                    

EXAMPLE 22

    ______________________________________                                        Zinc hyaluronate active ingredient                                                                          20.0   mg                                       Acrylic acid polymerisate     50     mg                                       Sodium hydroxide of 30% concentration                                                                       40     mg                                       Propylene glycol              1500   mg                                       Potassium sorbate             10     mg                                       Distilled water        up to  10.0   mg                                       ______________________________________                                    

IV. Creams and ointments for topical use

EXAMPLE 23

    ______________________________________                                        Zinc hyaluronate active ingredient                                                                          50     mg                                       Potassium sorbate             10     mg                                       Soft white bee wax            125    mg                                       Sorbitan oleate               150    mg                                       Cetyl stearyl alcohol         840    mg                                       Glyceryl monostearate         1100   mg                                       Propylene glycol              4750   mg                                       Distilled water        up to  10     g                                        ______________________________________                                    

EXAMPLE 24

    ______________________________________                                        Cobalt hyaluronate active ingredient                                                                        50     mg                                       Potassium sorbate             10     mg                                       Soft white bee wax            125    mg                                       Sorbitan oleate               150    mg                                       Cetyl stearyl alcohol         840    mg                                       Glyceryl monostearate         1100   mg                                       Propylene glycol              4750   mg                                       Distilled water        up to  10     g                                        ______________________________________                                    

EXAMPLE 25

    ______________________________________                                        Zinc hyaluronate active ingredient                                                                          50     mg                                       2-Phenoxyethanol              100    mg                                       Sodium lauryl sulfate         100    mg                                       Cetyl palmitate               400    mg                                       Stearin                       400    mg                                       Stearyl alcohol               450    mg                                       Cetyl alcohol                 450    mg                                       White vaseline                500    mg                                       Propylene glycol              550    mg                                       Glycerol                      600    mg                                       Distilled water        up to  10.0   g                                        ______________________________________                                    

EXAMPLE 26

    ______________________________________                                        Cobalt hyaluronate active ingredient                                                                        50     mg                                       2-Phenoxyethanol              100    mg                                       Sodium lauryl sulfate         100    mg                                       Cetyl palmitate               400    mg                                       Stearin                       400    mg                                       Stearyl alcohol               450    mg                                       Cetyl alcohol                 450    mg                                       White vaseline                500    mg                                       Propylene glycol              550    mg                                       Glycerol                      600    mg                                       Distilled water        up to  10     g                                        ______________________________________                                    

EXAMPLE 27

    ______________________________________                                        Zinc hyaluronate active ingredient                                                                          50.0   mg                                       Microcrystalline wax          250    mg                                       Propylene glycol              500    mg                                       Sorbitol                      400    mg                                       Wool wax (acetylated)         500    mg                                       White vaseline         up to  10     g                                        ______________________________________                                    

V. Compositions for the purification and cicatrization of purulentsounds and burns

EXAMPLE 28

    ______________________________________                                        Zinc hyaluronate active ingredient                                                                           10    mg                                       Potassium sorbate              1.0   mg                                       Hydrophilic colloidal silicon dioxide                                                                        50    mg                                       Sorbitol                up to  1     g                                        ______________________________________                                    

It is claimed:
 1. A method of treating a crural ulcer or decubitus ulcerin an afflicted patient which comprises the step of locallyadministering to said ulcer, a therapeutically effective amount of azinc hyaluronic acid complex with a stoichiometric composition, preparedthrough the interaction of equivalent amounts of sodium hyaluronate andzinc ions in aqueous solution, where the zinc is surrounded by fouroxygen atoms in the first coordination sphere, the length of the ZnObond distance is 199 pm whereas two carbon atoms are present in a longerdistance of 241 pm from the zinc atom.